PROJECT SUMMARY Intracellular cargo transport along microtubules is driven by kinesin and dynein motors that work antagonistically to precisely target cargo to specific cellular locations. Although the mechanisms underlying bidirectional transport have received considerable attention, a number of very fundamental questions remain, and a better quantitative understanding of molecular interactions and mechanochemistry underlying the regulation of bidirectional transport is essential for the field to move forward. In this work we will use DNA origami to create complexes with defined numbers of kinesin and dynein motors in vitro, and use Interfoerometric Scattering (iSCAT) microscopy to track them with 1 ms and 2 nm resolution. In parallel, we will model the transport using Brownian dynamics simulations to uncover the molecular-level details underlying the experimentally observed behavior. Our goal is to understand how the contrasting single-motor properties kinesin-1, -2, and -3 family motors manifest themselves in multi-motor teams, how roadblocks on microtubules alter bidirectional transport, and what role membrane fluidity plays in altering multi-motor transport properties. Aim 1 focuses on analyzing the movement of cargo transported by teams of similar and dissimilar kinesin motors to understand how differences in motor attachment rates and load-dependent detachment alter multi-motor behavior. Aim 2 will extend this to studying cargo functionalized with kinesin and activated mammalian cytoplasmic dynein complexes containing dynein, dynactin, and the activator BicD2. The switching behavior of different kinesins against their natural opponent will be of particular interest and will require high- resolution tracking in conjunction with experimentally constrained models. In Aim 3 we will attach motors to supported lipid bilayers and vesicles, measure their transport dynamics, and computationally model the underlying motor diffusion and bilayer deformation. We hypothesize that by acting as a ?shock absorber?, the fluid bilayer will reduce inter-motor forces, and that motor diffusion in the bilayer will lead to clustering that enhances motor performance in manner similar to integrins in focal adhesions. Although bidirectional transport can be described as a ?tug-of-war? between kinesin and dynein pulling in opposite directions, this model fails to account for a large body of experimental observations. This gulf between molecular-level understanding of motors in vitro and observed transport of cargo in cells must be bridged to understand the role of tau tangles in Alzheimers' disease, the deterioration of axonal transport in Huntington's and Parkinson's disease, and other neurodegenerative disorders that involve defects in microtubule-based bidirectional transport.